PROJECT SUMMARY In this proposal we aim to identify gene regulatory elements that permit the targeting and manipulation of brain circuit models of human brain function. Gaining genetic access to specific neuron populations in nontransgenic animals and humans would enable targeted circuit modulation for hypothesis testing and provide a means to evaluate the safety and efficacy of circuit modulation for the treatment of epilepsy and psychiatric disorders. Our approach capitalizes on our combined expertise in the development and maturation of brain cell-types and circuits (Gord Fishell), identification of CIS-regulatory elements that function across species (Jordane Dimidschstein) and AAV engineering combined with large-scale screening methods (Ben Deverman). Our efforts will benefit from an ongoing collaboration with John Reynolds at the Salk Institute on observation and manipulation of cortical circuits during complex visual perception tasks. This project will build upon success that we and others have had in identifying gene regulatory elements that enable cell type-restricted gene expression when used within recombinant adeno-associated virus (AAV) vectors. Identifying additional enhancer sequences that function in the context of the limited carrying capacity of AAV has been slow due to the limited success rate and low throughput nature of these efforts. Here we aim to apply a novel high-throughput screening approach for the rapid identification of a suite of enhancers that enable the study and manipulation of genetically defined cell types and circuits across species. Our preliminary data demonstrates that our enhancer identification strategy can yield novel and highly specific enhancers that restrict expression to target populations. In addition, we have demonstrated that it is possible to use the engineered AAV-PHP.eB capsid to screen enhancers across the brain with a single noninvasive injection. These successes have highlighted the need for more rapid and comprehensive assessment of putative enhancers. In the UH3 portion of this proposal we will examine the tolerance to neuronal activity manipulation within the target neuronal populations in several species. We will also apply the AAV-enhancer viruses for querying disease-related circuits using Rabies tracing in conjunction with optogenetics. This proposal will be transformative in devising methods to target and manipulate the brain activity of specific neuronal cell populations across species, including human cell-derived organoids.